1. Field of the Invention
The present invention relates to methods of medical treatment that use an external addition of pulses to body fluid channels in order to stimulate endothelial cells to release beneficial mediators. The circumferential shear stress within body fluid channels caused by these pulses stimulate the endothelial cells to produce mediators that become available for therapeutic and diagnostic purposes.
2. Description of the Related Art
It has become clear that the endothelial inner lining layer of the circulatory system, heart, lymphatics, interstitial spaces and bones of the body play a major role in health and disease through responses to shear stress from circulating fluids flowing across it or pulsating upon it. The recognition that vascular endothelium is a highly active metabolic organ came with the discovery that it actively liberated nitric oxide, a powerful relaxant of vascular smooth muscle as a function of shear stress and pulse frequency across the endothelial surface.
Nitric oxide is synthesized in many cell types by the conversion of L-arginine, a naturally occurring amino acid, to L-citrulline by three distinct nitric oxide synthases (NOSs) enzymes. Sustained high levels of nitric oxide are produced by macrophage or smooth muscle inducible NOS (iNOS) only after induction by certain cytokinines. Both neuronal (nNOS) and endothelial (eNOS) are calcium-dependent and produce low levels of nitric oxide constitutively. eNOS acts in the presence of nicotinamide-adenine-dinucleotide phosphate (NADPH), Ca++2/calmodulin and tetrahydrobiopterin to oxidize the L-arginine and form nitric oxide and L-citrulline. Nitric oxide stimulates soluble guanylate cyclase in the underlying vascular smooth muscle that leads to elevation of cyclic guanosine monophosphate (cGMP) with consequent vascular relaxation.
Release of nitric oxide causes endothelium dependent vasodilation. The wrapping of a tourniquet around the upper arm for a few minutes followed by quick removal allows increased blood to flow into the lower arm, a maneuver termed reactive hyperemia; this causes local increase in flow shear stress. Increased pulse frequency acting upon the endothelium is achieved during exercise (also accompanied by increased flow shear stress) or with electrical pacing of the heart.
On the other hand, endothelin-1, a powerful vasoconstrictor substance, is released in higher amounts when shear stress diminishes. Increase of nitric oxide release suppresses endothelin-1. This interplay between these two mediators helps stabilize blood pressure levels. L-arginine, a naturally occurring amino acid, is the substrate for the enzyme in endothelium, eNOS, which converts it to nitric oxide where it acts on smooth muscle and is metabolized into serum nitrite and other NO-compounds within 5 to 10 seconds. Circulating nitrosylated compounds are agents that take up nitric oxide to a lesser extent and slowly release nitric oxide over prolonged time periods.
Prostacyclin is liberated from endothelium by shear stress and relaxes vascular smooth muscle as an endothelium independent vasodilator. In most blood vessels, the contribution of prostacyclin to endothelial-dependent vasodilation is small and its effect is additive to nitric oxide. However, in terms of preventing platelet aggregation, leukocyte adhesion to endothelium, and susceptibility to thrombosis, the action of prostacyclin and nitric oxide are synergistic. Nitric oxide has an inhibitory effect on prostacyclin production under shear stress but vessel homeostasis is maintained through an increase in prostacyclin production when nitric oxide synthesis is impaired in endothelial cells as in atherosclerosis.
Shear stress independent of perfusion pressure increases gene expression of prostacyclin synthesis-related enzymes cyclooxygenases (COX-1 and COX-2), prostacyclin synthase (PGS), and thromboxane synthase (TXS) and their metabolites prostaglandin (PGI(2)) and thromboxane A(2) (TXA(2)) in endothelium of intact conduit vessels.
In the epicardial coronary arteries, shear stress causes release of endothelial-dependent hyperpolarizing factor (EDHF). Here, its formation plays a much greater role than either nitric oxide or prostacyclin, since in this vascular bed, endothelium-dependent vasodilation is only marginally attenuated by combined inhibition of nitric oxide synthase and cyclooxygenase. In the coronary circulation, EDHF displays the characteristics of a cytochrome P450-dependent arachnoidonic acid metabolite. Ultimately, EDHF acts through the nitric oxide-L-arginine pathway. Sinusoidal pressure oscillations (from 40 to 50 mm Hg, 4 minutes, 1.5 Hz) leads to simultaneous oscillations in the external diameter of isolated coronary artery segments, the amplitude of which were decreased by iberiotoxin and apamin and also by endothelial denudation. Thus, continuous release of EDHF may contribute to the adjustment of an adequate vascular compliance and to the control of coronary blood flow.
Tissue plasminogen activator (t-PA) is released from vascular endothelium through shear stress. Further, shear stress is a potent fluid mechanical stimulus for upregulation of the intracellular storage pool of t-PA in the vascular wall. Shear stress effect is associated with an increased t-PA gene expression. t-PA is measurable in plasma and therefore also is a marker of endothelial function. Muscarinic agents such as acetylcholine and methacholine release tissue plasminogen activator in the forearm circulation of normal subjects. In patients with hypertension, acetylcholine does not change flow and net release and concentration gradients of t-PA, but increases blood flow in normal subjects indicating that vasodilatation by increasing fluid shear stress induces t-PA release with normally functioning vascular endothelium. Marked t-PA release occurs in response to isoproterenol, a beta-adrenergic agonist that acts through the nitric oxide-L-arginine pathway. This effect is independent of the effects of shear stress due to increased blood flow because nitroprusside, an endothelium-independent vasodilator induces similar increases in blood flow without causing t-PA release. Possibly, circumferential shear stress is a more potent stimulus to t-PA release than tangential shear stress.
Elevated intraluminal pressure downregulates t-PA gene and protein expression and inhibits its release from the endothelium independently of shear stress. The defective capacity for stimulated t-PA release that is demonstrable in patients with systemic essential hypertension might thus be an effect of the elevated intraluminal pressure per se.
Activator protein-1 (AP-1) is composed of c-fos/c-jun hererodimers or c-jun/c-jun homodiamers. This is released from the vascular endothelium with shear stress and/or circumferential pulses. The AP-1 transcription factor family is important in the transcription of several genes, e.g., monocyte chemotactic protein-1 (MCP-1) and the vascular cell adhesion molecule-1. Endothelial cells subjected to disturbed laminar shear stress exhibit increased levels of nuclear localized NF-kappaB, Egr-1, c-Jun, and c-Fos, compared with cells exposed to uniform laminar shear stress or maintained under static conditions. In addition, individual cells display a heterogeneity in responsiveness to disturbed flow, as measured by the amount of NF-kappaB, Egr-1, c-Jun, and c-Fos in their nuclei. This differential regulation of transcription factor expression by disturbed versus uniform laminar shear stress indicates that regional differences in blood flow patterns in vivo-in particular, the occurrence of spatial shear stress gradients-may represent important local modulators of endothelial gene expression at anatomic sites predisposed for atherosclerotic development.
Shear stress acts at the apical cell surface to deform cells in the direction of blood flow; wall distention from cycical strain tends to deform cells in all directions. The shear stress response differs, at least partly, from the cyclical strain response, suggesting that cytoskeletal strain alone cannot explain it. Acute shear stress in vitro elicits rapid cytoskeletal remodeling and activates signaling cascades in endothelial cells, with the consequent acute release of nitric oxide, prostacyclin, t-PA and EDHF; activation of transcription factors nuclear factor (NF), kappaB, c-fos, c-jun and SP-1; and transcriptional activation of genes, including ICAM-1, MCP-1, tissue factor, platelet-derived growth factor-B (PDGF-B), transforming growth factor (TGF)-beta1, cyclooxygenase-II, and endothelial nitric oxide synthase (eNOS). Thus, the forces acting upon the vascular endothelium from laminar shear stress and pulsations cause release of a myriad of active agents.
However beneficial the effects of these mediators may be, it is difficult to dose a patient with them. For example, nitric oxide is currently administered as a gas in concentrations from 20 to 80 ppm. Since nitric oxide rapidly combines with hemoglobin competing for oxygenation, it can be considered a toxic gas. Its concentration must be carefully monitored. It undergoes rapid degradation in the pulmonary circulation and has no systemic effects. There are some benefits to inhaled nitric oxide: the pulmonary vascular hypertensive response to acute hypoxia is abolished by NO inhalation therapy. However, the pulmonary hypertensive response to aspirated meconium is only partially reversed by inhaled nitric oxide therapy despite improvements in oxygenation.
Besides inhaled nitric oxide, there are nitric oxide donors. There are substantial differences among the diverse classes of nitric oxide donors including relative importance of nonenzymatic versus enzymatic pathways for NO release, existence of competing metabolic events and the identity of the actual NO-generating enzyme systems. For example, organic nitrates are predominantly venodilators that selectively reduce cardiac preload whereas sodium nitroprusside relaxes arteries and veins equally. The sensitivity of soluble guanylate cyclase to NO donors might be regulated by the ambient concentration of NO, with increased local nitric oxide downregulation of the dilator response to NO donors.
Compounds such as nitroglycerin, nitroprusside and other organic nitrate compounds release nitric oxide through enzymatic degradation and act directly on vascular smooth muscle to cause vasodilation. These compounds are designated endothelium independent vasodilators since they relax vascular smooth muscle even though vascular endothelium may be dysfunctional or destroyed at a given site of action. However, enzymatic conversion may be incomplete at different sites or other vasoactive compounds may be formed leading to different actions on different vascular beds. This may cause drug tolerance, less effectiveness for a given dose of an organic nitrate. Tolerance to continuously administered transdermal nitroglycerin can develop with 48 hours.
Thus, there is a need for a method of providing beneficial mediators (such as nitric oxide, prostacyclin, endothelial dependent hyperpolarizing factor (EDHF), and tissue plasminogen activator (t-PA)) that are released by the endothelium, in order to aid in the treatment and diagnosis of various diseases, conditions, and injuries. Furthermore, the method must avoid the dosing problems of previous treatments.